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Abstract:

The present invention provides vectors that contain and express in vivo
or in vitro one or more Hendra virus polypeptides or antigens that elicit
an immune response in animal or human against Hendra virus and Nipah
virus, compositions comprising said vectors and/or Hendra virus
polypeptides, methods of vaccination against Hendra virus and Nipah
virus, and kits for use with such methods and compositions.

Claims:

1. A composition comprising one or more expression vectors, wherein the
vector comprises a polynucleotide encoding one or more polypeptide
selected from the group consisting of a Hendra virus G polypeptide, a
variant or fragment of the Hendra virus G polypeptide, a Hendra virus F
polypeptide, a variant or fragment of the Hendra virus F polypeptide, and
a mixture thereof.

2. The composition of claim 1, wherein the vector comprises a first
polynucleotide encoding a Hendra virus G polypeptide and a second
polynucleotide encoding a Hendra virus F polypeptide.

3. The composition of claim 1, wherein the composition comprises a first
expression vector comprising a polynucleotide encoding a Hendra virus G
polypeptide and a second expression vector comprising a polynucleotide
encoding a Hendra virus F polypeptide.

4. The composition of claim 1 further comprising one or more additional
antigens.

5. The composition of claim 4, wherein the additional antigens are Nipah
antigens.

6. The composition of claim 2, wherein the first polynucleotide encodes a
Hendra virus G polypeptide having at least 80% sequence identity to the
sequence as set forth in SEQ ID NO: 3, and wherein the second
polynucleotide encodes a Hendra virus F polypeptide having at least 80%
sequence identity to the sequence as set forth in SEQ ID NO: 6.

7. The composition of claim 1, wherein the polynucleotide encodes a
Hendra virus G polypeptide having at least 80% sequence identity to the
sequence as set forth in SEQ ID NO:3.

8. The composition of claim 1, wherein the polynucleotide encodes a
Hendra virus F polypeptide having at least 80% sequence identity to the
sequence as set forth in SEQ ID NO:6.

9. The composition of claim 1, wherein the polynucleotide has at least
70% sequence identity to the sequence as set forth in SEQ ID NO:1, 2, 4,
or 5.

10. The composition of claim 1, wherein the vector is a poxvirus.

11. The composition of claim 1, wherein the composition further comprises
a pharmaceutically or veterinary acceptable vehicle, adjuvant, diluent or
excipient.

12. A vector comprising one or more polynucleotide encoding one or more
polypeptide selected from the group consisting of a Hendra virus G
polypeptide, a variant or fragment of the Hendra virus G, a Hendra virus
F polypeptide, a variant or fragment of the Hendra virus F polypeptide,
and a mixture thereof.

13. The vector of claim 12, wherein the polynucleotide encodes a Hendra
virus G polypeptide having at least 80% sequence identity to the sequence
as set forth in SEQ ID NO:3.

14. The vector of claim 12, wherein the polynucleotide encodes a Hendra
virus F polypeptide having at least 80% sequence identity to the sequence
as set forth in SEQ ID NO:6.

15. The vector of claim 12, wherein the vector comprises a first
polynucleotide encoding a Hendra virus G polypeptide and a second
polynucleotide encoding a Hendra virus F polypeptide.

16. The vector of claim 15, wherein the first polynucleotide encodes a
Hendra virus G polypeptide having at least 80% sequence identity to the
sequence as set forth in SEQ ID NO: 3, and wherein the second
polynucleotide encodes a Hendra virus F polypeptide having at least 80%
sequence identity to the sequence as set forth in SEQ ID NO: 6.

17. The vector of claim 12, wherein the polynucleotide is operably linked
to a promoter.

18. The vector of claim 12, wherein the vector is a poxvirus.

19. A method of vaccinating an animal comprising at least one
administration of the composition of claim 1 or vector of claim 12.

Description:

[0002] The present invention relates to formulations for combating Hendra
virus and Nipah virus in animals. Specifically, the present invention
provides vectors that contain and express in vivo or in vitro Hendra
virus F and G antigens that elicit an immune response in animals and
human against Hendra virus and Nipah virus, including compositions
comprising said vectors, methods of vaccination against Hendra virus and
Nipah virus, and kits for use with such methods and compositions. The
present invention also provides vectors that contain and express in vivo
or in vitro Hendra F or G protein that elicit an immune response in
animals against Hendra virus and Nipah, and compositions comprising said
vectors.

BACKGROUND OF THE INVENTION

[0003] Hendra virus is the source of a recently emerging disease in
animals and human. Hendra virus was first recognized in September 1994
after an outbreak of respiratory illness among twenty horses and two
humans in Hendra, Queensland, Australia (Selvey L A, et al., Med J
Australia 1995, 162:642-5). Thirteen horses and one human died. In 1995,
a second unrelated outbreak was identified that had occurred in August
1994 in Mackay, Queensland, in which two horses died and one human became
infected (Hooper P T, et al., Australian Vet J 1996; 74:244-5; Rogers R
J, et al., Australia Vet J 1996; 74:243-4). Four of the seven people who
contracted the virus from infected horses have died since the disease
first emerged in 1994. The fatality rate has been reported at more than
70% in horses and 50% in humans.

[0004] Nipah virus is a member of the Paramyxoviridae family and is
related to the Hendra virus (formerly called equine morbillivirus). The
Nipah virus was initially isolated in 1999 upon examining samples from an
outbreak of encephalitis and respiratory illness among adult men in
Malaysia and Singapore (see, e.g., Chua et al., Lancet. 1999, 354
(9186):1257-9 and Paton et al., Lancet. 1999 Oct. 9; 354(9186):1253-6).
The host for Nipah virus is still unknown, but flying foxes (bats of the
Pteropus genus) are suspected to be the natural host. Infection with
Nipah virus in humans has been associated with an encephalitis
characterized by fever and drowsiness and more serious central nerve
system disease, such as coma, seizures and inability to maintain
breathing (see, e.g., Lee et al., Ann Neurol. 1999 September;
46(3):428-32). Illness with Nipah virus begins with 3-14 days of fever
and headache, followed by drowsiness and disorientation characterized by
mental confusion. These signs and symptoms can progress to coma within
24-48 hours. Some patients have had a respiratory illness during the
early part of their infections. Serious nerve disease with Nipah virus
encephalitis has been marked by some sequelae, such as persistent
convulsions and personality changes. During the Nipah virus disease
outbreak in 1998-1999, about 40% of the patients with serious nerve
disease who entered hospitals died from the illness (see, e.g., Lam &
Chua, Clin Infect Dis. 2002 May 1; 34 Suppl 2:S48-51).

[0005] Hendra virus, like the majority of other paramyxoviruses, possess
two surface glycoproteins, a fusion protein (F) and an attachment protein
(G), that are involved in promotion of fusion between the viral membrane
and the membrane of the target host cell. Hendra and Nipah viruses
require both their attachment and fusion proteins to initiate membrane
fusion (Bossart et al., J Virol. 2002; 76:11186-98). Various studies were
conducted to understand the functions of the G and F proteins in virus
infection. A soluble G glycoprotein of Hendra virus was constructed and
showed the capability to bind to Hedra virus and Nipah virus
infection-permissive cells (Bossart et al., J Virol. 2005; 79:6690-6702).
Monoclonal antibodies specific for the Nipah virus fusion protein were
shown to neutralize Hedra virus in vitro and protected hamsters from
Hendra virus (Guillaume et al., Virology 2009; 387:459-465). A
recombinant soluble Hendra G protein in CpG adjuvant was evaluated in a
cat model (McEachern et al., Vaccine 2008; 26:3842-3852).

[0006] Currently there is no licensed Hendra vaccine. Therefore, there is
a general need for a Hendra vaccine for the protection against Hendra
virus and Nipah virus infection, prevention of the disease in animals and
human and prevention of spreading of the virus to uninfected animals or
human.

[0007] The invention provides a solution for optimizing the immunological
and efficacious effect of Hendra virus vaccine while retaining high
safety for the vaccinated animals.

SUMMARY OF THE INVENTION

[0008] An object of this invention can be any one or all of providing
recombinant vectors or viruses as well as methods for making such
viruses, and providing compositions and/or vaccines as well as methods
for treatment and prophylaxis of infection by Hendra virus or Nipah
virus.

[0009] The invention provides a recombinant vector, such as a recombinant
virus, that contains and expresses at least one exogenous nucleic acid
molecule and, the at least one exogenous nucleic acid molecule may
comprise a nucleic acid molecule encoding an immunogen or epitope of
interest from Hendra virus, such as F or G or a combination thereof.

[0010] The invention further provides compositions or vaccines comprising
such an expression vector or the expression product(s) of such an
expression vector. The compositions or vaccines may comprise two or more
such expression vectors or the expression product(s) of such expression
vectors. The invention further relates to a vaccine or composition which
may comprise one or more aforementioned recombinant or expression vector
a pharmaceutically or veterinarily acceptable carrier, excipient,
adjuvant, or vehicle, and additionally one or more antigens. The
additional antigen(s) may be Nipah virus antigen(s).

[0011] The invention further provides methods for inducing an
immunological (or immunogenic) or protective response against Hendra
virus or Nipah virus, as well as methods for preventing or treating the
disease state(s) caused by Hendra virus or Nipah virus, comprising
administering the expression vector or an expression product of the
expression vector, or a composition comprising the expression vector, or
a composition comprising an expression product of the expression vector.

[0012] The invention relates to expression products from the virus as well
as antibodies generated from the expression products or the expression
thereof in vivo and uses for such products and antibodies, e.g., in
diagnostic applications. The invention also relates to a method of
hyperimmunizing horses to induce polyclonal antibodies for serotherapy in
animals and humans comprising at least one administration of the
composition or vector of the present invention.

[0013] These and other embodiments are disclosed or are obvious from and
encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description, given by way of example, but
not intended to limit the invention solely to the specific embodiments
described, may best be understood in conjunction with the accompanying
drawings, in which:

[0015] FIG. 1 is the table showing the SEQ ID NO assigned to the
respective DNA and Protein sequences.

[0027] Compositions comprising one or more expression vector(s) comprising
one or more polynucleotide(s) encoding one or more Hendra virus
antigen(s), polypeptide(s) and fragments and variants thereof that elicit
an immunogenic response in an animal or human are provided. The
expression vector comprising the polynucleotide encoding Hendra virus
antigen(s) or polypeptide(s) or fragments or variants may be formulated
into vaccines or pharmaceutical compositions and used to elicit or
stimulate a protective response in an animal or human. In one embodiment
the Hendra virus antigen or polypeptide is a Hendra virus fusion protein
(F), a Hendra virus attachment protein (G), or active fragment or variant
thereof.

[0028] It is recognized that the polypeptides of the invention may be full
length polypeptides or active fragments or variants thereof. By "active
fragments" or "active variants" is intended that the fragments or
variants retain the antigenic nature of the polypeptide. Thus, the
present invention encompasses any Hendra virus polypeptide, antigen,
epitope or immunogen that elicits an immunogenic response in an animal.
The Hendra virus polypeptide, antigen, epitope or immunogen may be any
Hendra virus polypeptide, antigen, epitope or immunogen, such as, but not
limited to, a protein, peptide or fragment or variant thereof, that
elicits, induces or stimulates a response in an animal.

[0029] A particular Hendra virus polypeptide of interest is Hendra virus
fusion protein (F) and Hendra virus attachment protein (G). It is further
recognized that precursors of any of these antigens can be used. The
antigenic polypeptides of the invention are capable of protecting against
Hendra virus. That is, they are capable of stimulating an immune response
in an animal or human.

[0030] The term "antigen" or "immunogen" means a substance that induces a
specific immune response in a host animal. The antigen may comprise a
whole organism, killed, attenuated or live; a subunit or portion of an
organism; a recombinant vector containing an insert with immunogenic
properties; a piece or fragment of DNA capable of inducing an immune
response upon presentation to a host animal; a polypeptide, an epitope, a
hapten, or any combination thereof. Alternately, the immunogen or antigen
may comprise a toxin or antitoxin.

[0031] The terms "protein", "peptide", "polypeptide" and "polypeptide
fragment" are used interchangeably herein to refer to polymers of amino
acid residues of any length. The polymer can be linear or branched, it
may comprise modified amino acids or amino acid analogs, and it may be
interrupted by chemical moieties other than amino acids. The terms also
encompass an amino acid polymer that has been modified naturally or by
intervention; for example disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other manipulation or
modification, such as conjugation with a labeling or bioactive component.

[0032] The term "Hendra virus polypeptide or antigen" refers to any
antigen or polypeptide identified in any Hendra virus strain. The antigen
or polypeptide may be native to the particular Hendra virus strain. The
antigen or polypeptide may be optimized from its native form. Hendra
virus polypeptide or antigen include, for example, fusion protein (F),
attachment protein (G), and Nucleocapsid (N) protein.

[0033] The term "immunogenic or antigenic polypeptide" as used herein
includes polypeptides that are immunologically active in the sense that
once administered to the host, it is able to evoke an immune response of
the humoral and/or cellular type directed against the protein. Preferably
the protein fragment is such that it has substantially the same
immunological activity as the total protein. Thus, a protein fragment
according to the invention comprises or consists essentially of or
consists of at least one epitope or antigenic determinant. An
"immunogenic" protein or polypeptide, as used herein, includes the
full-length sequence of the protein, analogs thereof, or immunogenic
fragments thereof. By "immunogenic fragment" is meant a fragment of a
protein which includes one or more epitopes and thus elicits the
immunological response described above. Such fragments can be identified
using any number of epitope mapping techniques well known in the art.
See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes may be
determined by e.g., concurrently synthesizing large numbers of peptides
on solid supports, the peptides corresponding to portions of the protein
molecule, and reacting the peptides with antibodies while the peptides
are still attached to the supports. Such techniques are known in the art
and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al., 1984;
Geysen et al., 1986. Similarly, conformational epitopes are readily
identified by determining spatial conformation of amino acids such as by,
e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance.
See, e.g., Epitope Mapping Protocols, supra.

[0034] As discussed herein, the invention encompasses active fragments and
variants of the antigenic polypeptide. Thus, the term "immunogenic or
antigenic polypeptide" further contemplates deletions, additions and
substitutions to the sequence, so long as the polypeptide functions to
produce an immunological response as defined herein. The term
"conservative variation" denotes the replacement of an amino acid residue
by another biologically similar residue, or the replacement of a
nucleotide in a nucleic acid sequence such that the encoded amino acid
residue does not change or is another biologically similar residue. In
this regard, particularly preferred substitutions will generally be
conservative in nature, i.e., those substitutions that take place within
a family of amino acids. For example, amino acids are generally divided
into four families: (1) acidic--aspartate and glutamate; (2)
basic--lysine, arginine, histidine; (3) non-polar--alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and
(4) uncharged polar--glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified as aromatic amino acids. Examples of conservative
variations include the substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another hydrophobic
residue, or the substitution of one polar residue for another polar
residue, such as the substitution of arginine for lysine, glutamic acid
for aspartic acid, or glutamine for asparagine, and the like; or a
similar conservative replacement of an amino acid with a structurally
related amino acid that will not have a major effect on the biological
activity. Proteins having substantially the same amino acid sequence as
the reference molecule but possessing minor amino acid substitutions that
do not substantially affect the immunogenicity of the protein are,
therefore, within the definition of the reference polypeptide. All of the
polypeptides produced by these modifications are included herein. The
term "conservative variation" also includes the use of a substituted
amino acid in place of an unsubstituted parent amino acid provided that
antibodies raised to the substituted polypeptide also immunoreact with
the unsubstituted polypeptide.

[0035] The term "epitope" refers to the site on an antigen or hapten to
which specific B cells and/or T cells respond. The term is also used
interchangeably with "antigenic determinant" or "antigenic determinant
site". Antibodies that recognize the same epitope can be identified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen.

[0036] An "immunological response" to a composition or vaccine is the
development in the host of a cellular and/or antibody-mediated immune
response to a composition or vaccine of interest. Usually, an
"immunological response" includes but is not limited to one or more of
the following effects: the production of antibodies, B cells, helper T
cells, and/or cytotoxic T cells, directed specifically to an antigen or
antigens included in the composition or vaccine of interest. Preferably,
the host will display either a therapeutic or protective immunological
response such that resistance to new infection will be enhanced and/or
the clinical severity of the disease reduced. Such protection will be
demonstrated by either a reduction or lack of symptoms normally displayed
by an infected host, a quicker recovery time and/or a lowered viral titer
in the infected host.

[0038] Unless otherwise explained, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs. The singular terms
"a", "an", and "the" include plural referents unless context clearly
indicates otherwise. Similarly, the word "or" is intended to include
"and" unless the context clearly indicate otherwise.

[0039] It is noted that in this disclosure and particularly in the claims
and/or paragraphs, terms such as "comprises", "comprised", "comprising"
and the like can have the meaning attributed to it in U.S. patent law;
e.g., they can mean "includes", "included", "including", and the like;
and that terms such as "consisting essentially of" and "consists
essentially of" have the meaning ascribed to them in U.S. patent law,
e.g., they allow for elements not explicitly recited, but exclude
elements that are found in the prior art or that affect a basic or novel
characteristic of the invention.

Compositions

[0040] The present invention relates to a Hendra virus recombinant vaccine
or composition which may comprise at least one recombinant or expression
vector comprising one or more polynucleotide(s) encoding one or more
Hendra virus polypeptide, antigen, epitope or immunogen. The vaccine or
composition may further comprise a pharmaceutically or veterinarily
acceptable carrier, excipient, adjuvant, or vehicle. The Hendra virus
polypeptide, antigen, epitope or immunogen may be any Hendra virus
polypeptide, antigen, epitope or immunogen, such as, but not limited to,
a protein, peptide or fragment thereof, that elicits, induces or
stimulates a response in an animal.

[0041] In another embodiment, the pharmaceutically or veterinarily
acceptable carrier, excipient, adjuvant, or vehicle may be a water-in-oil
emulsion. In yet another embodiment, the water-in-oil emulsion may be a
water/oil/water (W/O/W) triple emulsion. In yet another embodiment, the
pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant,
or vehicle may be an oil-in-water emulsion. In another embodiment, the
pharmaceutically or veterinarily acceptable carriers, excipients,
adjuvants, or vehicles may be polymers of acrylic or methacrylic acid,
maleic anhydride and alkenyl derivative polymers.

[0042] In an embodiment, the Hendra virus polypeptide, antigen or fragment
or variant thereof may be a Hendra virus F polypeptide or fragment or
variant thereof. In an aspect of this embodiment, the Hendra virus F
polypeptide or fragment or variant thereof is a recombinant polypeptide
produced by a Hendra virus F gene. In another aspect of this embodiment,
the Hendra virus F gene has at least 70% identity to the sequence as set
forth in SEQ ID NO: 4 or 5. In another aspect of this embodiment, the
Hendra virus F polypeptide or fragment or variant thereof has at least
80% identity to the sequence as set forth in SEQ ID NO: 6.

[0043] In another embodiment, the Hendra virus polypeptide, antigen or
fragment or variant thereof may be a Hendra virus G polypeptide or
fragment or variant thereof. In an aspect of this embodiment, the Hendra
virus G polypeptide or fragment or variant thereof is a recombinant
polypeptide produced by a Hendra virus G gene. In another aspect of this
embodiment, the Hendra virus G gene has at least 70% identity to the
sequence as set forth in SEQ ID NO: 1 or 2. In another aspect of this
embodiment, the Hendra virus G polypeptide or fragment or variant thereof
has at least 80% identity to the sequence as set forth in SEQ ID NO: 3.

[0044] Synthetic antigens are also included within the definition, for
example, polyepitopes, flanking epitopes, and other recombinant or
synthetically derived antigens. Immunogenic fragments, for purposes of
the present invention, will usually include at least about 3 amino acids,
at least about 5 amino acids, at least about 10-15 amino acids, or about
15-25 amino acids or more amino acids, of the molecule. There is no
critical upper limit to the length of the fragment, which could comprise
nearly the full-length of the protein sequence, or even a fusion protein
comprising at least one epitope of the protein.

[0045] Accordingly, a minimum structure of a polynucleotide expressing an
epitope is that it comprises or consists essentially of or consists of
nucleotides encoding an epitope or antigenic determinant of a Hendra
virus polypeptide. A polynucleotide encoding a fragment of a Hendra virus
polypeptide may comprise or consist essentially of or consist of a
minimum of 15 nucleotides, about 30-45 nucleotides, about 45-75, or at
least 75, 87 or 150 consecutive or contiguous nucleotides of the sequence
encoding the polypeptide. Epitope determination procedures, such as,
generating overlapping peptide libraries (Hemmer et al., 1998), Pepscan
(Geysen et al., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989;
Geysen, 1990; Multipin® Peptide Synthesis Kits de Chiron) and
algorithms (De Groot et al., 1999; PCT/US2004/022605) can be used in the
practice of the invention.

[0046] The term "nucleic acid" and "polynucleotide" refers to RNA or DNA
that is linear or branched, single or double stranded, or a hybrid
thereof. The term also encompasses RNA/DNA hybrids. The following are
non-limiting examples of polynucleotides: a gene or gene fragment, exons,
introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes and primers.
A polynucleotide may comprise modified nucleotides, such as methylated
nucleotides and nucleotide analogs, uracyl, other sugars and linking
groups such as fluororibose and thiolate, and nucleotide branches. The
sequence of nucleotides may be further modified after polymerization,
such as by conjugation, with a labeling component. Other types of
modifications included in this definition are caps, substitution of one
or more of the naturally occurring nucleotides with an analog, and
introduction of means for attaching the polynucleotide to proteins, metal
ions, labeling components, other polynucleotides or solid support. The
polynucleotides can be obtained by chemical synthesis or derived from a
microorganism.

[0047] The term "gene" is used broadly to refer to any segment of
polynucleotide associated with a biological function. Thus, genes include
introns and exons as in genomic sequence, or just the coding sequences as
in cDNAs and/or the regulatory sequences required for their expression.
For example, gene also refers to a nucleic acid fragment that expresses
mRNA or functional RNA, or encodes a specific protein, and which includes
regulatory sequences.

[0048] An "isolated" biological component (such as a nucleic acid or
protein or organelle) refers to a component that has been substantially
separated or purified away from other biological components in the cell
of the organism in which the component naturally occurs, for instance,
other chromosomal and extra-chromosomal DNA and RNA, proteins, and
organelles. Nucleic acids and proteins that have been "isolated" include
nucleic acids and proteins purified by standard purification methods. The
term also embraces nucleic acids and proteins prepared by recombinant
technology as well as chemical synthesis.

[0049] The term "purified" as used herein does not require absolute
purity; rather, it is intended as a relative term. Thus, for example, a
partially purified polypeptide preparation is one in which the
polypeptide is more enriched than the polypeptide is in its natural
environment. That is the polypeptide is separated from cellular
components. By "substantially purified" is intended that at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or
more of the cellular components or materials have been removed. Likewise,
a polypeptide may be partially purified. By "partially purified" is
intended that less than 60% of the cellular components or material is
removed. The same applies to polynucleotides. The polypeptides disclosed
herein can be purified by any of the means known in the art.

[0050] Moreover, homologs of Hendra virus F or G polypeptides are intended
to be within the scope of the present invention. As used herein, the term
"homologs" includes orthologs, analogs and paralogs. The term "anologs"
refers to two polynucleotides or polypeptides that have the same or
similar function, but that have evolved separately in unrelated
organisms. The term "orthologs" refers to two polynucleotides or
polypeptides from different species, but that have evolved from a common
ancestral gene by speciation. Normally, orthologs encode polypeptides
having the same or similar functions. The term "paralogs" refers to two
polynucleotides or polypeptides that are related by duplication within a
genome. Paralogs usually have different functions, but these functions
may be related. For example, analogs, orthologs, and paralogs of a
wild-type Hendra virus polypeptide can differ from the wild-type Hendra
virus polypeptide by post-translational modifications, by amino acid
sequence differences, or by both. In particular, homologs of the
invention will generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%,
96%, 97%, 98% , 99% sequence identity, with all or part of the wild-type
Hendra virus polypeptide or polynucleotide sequences, and will exhibit a
similar function.

[0051] In one embodiment, the present invention provides an expression
vector comprising one or more polynucleotides encoding one or more
polypeptides having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity
to a polypeptide having a sequence as set forth in SEQ ID NO: 3 or 6. In
another embodiment, the present invention provides fragments and variants
of the Hendra virus F or G polypeptides identified above (SEQ ID NO: 3,
6) which may readily be prepared by one of skill in the art using
well-known molecular biology techniques. Variants are homologous
polypeptides having amino acid sequences at least 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences as set
forth in SEQ ID NO: 3 or 6.

[0052] Variants include allelic variants. The term "allelic variant"
refers to a polynucleotide or a polypeptide containing polymorphisms that
lead to changes in the amino acid sequences of a protein and that exist
within a natural population (e.g., a virus species or variety). Such
natural allelic variations can typically result in 1-5% variance in a
polynucleotide or a polypeptide. Allelic variants can be identified by
sequencing the nucleic acid sequence of interest in a number of different
species, which can be readily carried out by using hybridization probes
to identify the same genetic locus in those species. Any and all such
nucleic acid variations and resulting amino acid polymorphisms or
variations that are the result of natural allelic variation and that do
not alter the functional activity of gene of interest, are intended to be
within the scope of the invention.

[0053] As used herein, the term "derivative" or "variant" refers to a
polypeptide, or a nucleic acid encoding a polypeptide, that has one or
more conservative amino acid variations or other minor modifications such
that (1) the corresponding polypeptide has substantially equivalent
function when compared to the wild type polypeptide or (2) an antibody
raised against the polypeptide is immunoreactive with the wild-type
polypeptide. These variants or derivatives include polypeptides having
minor modifications of the Hendra virus polypeptide primary amino acid
sequences that may result in peptides which have substantially equivalent
activity as compared to the unmodified counterpart polypeptide. Such
modifications may be deliberate, as by site-directed mutagenesis, or may
be spontaneous. The term "variant" further contemplates deletions,
additions and substitutions to the sequence, so long as the polypeptide
functions to produce an immunological response as defined herein.

[0054] An immunogenic fragment of a Hendra virus polypeptide includes at
least 8, 10, 13, 14, 15, or 20 consecutive amino acids, at least 21 amino
acids, at least 23 amino acids, at least 25 amino acids, or at least 30
amino acids of a Hendra virus polypeptide having a sequence as set forth
in SEQ ID NO: 3, 6, or variants thereof.

[0055] In another aspect, the present invention provides an expression
vector comprising a polynucleotide encoding a Hendra virus F polypeptide,
such as a polynucleotide encoding a polypeptide having a sequence as set
forth in SEQ ID NO: 6. In yet another aspect, the present invention
provides an expression vector comprising a polynucleotide encoding a
polypeptide having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity
to a polypeptide having a sequence as set forth in SEQ ID NO: 6, or a
conservative variant, an allelic variant, a homolog or an immunogenic
fragment comprising at least eight or at east ten consecutive amino acids
of one of these polypeptides, or a combination of these polypeptides.

[0056] In yet another aspect, the present invention provides an expression
vector comprising a polynucleotide encoding a Hendra virus G polypeptide,
such as a polynucleotide encoding a polypeptide having a sequence as set
forth in SEQ ID NO: 3. In yet another aspect, the present invention
provides an expression vector comprising a polynucleotide encoding a
polypeptide having at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity
to a polypeptide having a sequence as set forth in SEQ ID NO: 3, or a
conservative variant, an allelic variant, a homolog or an immunogenic
fragment comprising at least eight or at east ten consecutive amino acids
of one of these polypeptides, or a combination of these polypeptides.

[0057] In yet another aspect, the present invention provides an expression
vector comprising two polynucleotides encoding a Hendra virus F
polypeptide, such as a polynucleotide encoding a polypeptide having a
sequence as set forth in SEQ ID NO: 6 and a Hendra virus G polypeptide,
such as a polynucleotide encoding a polypeptide having a sequence as set
forth in SEQ ID NO: 3.

[0058] In one embodiment the polynucleotide of the present invention
includes a polynucleotide having a nucleotide sequence as set forth in
SEQ ID NO: 1, 2, 4, 5, or a variant thereof. In another embodiment, the
polynucleotide of the present invention includes a polynucleotide having
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 95%, 96%, 97%, 98% or 99% sequence identity to one of
a polynucleotide having a sequence as set forth in SEQ ID NO: 1, 2, 4, 5,
or a variant thereof.

[0059] The polynucleotides of the disclosure include sequences that are
degenerate as a result of the genetic code, e.g., optimized codon usage
for a specific host. As used herein, "optimized" refers to a
polynucleotide that is genetically engineered to increase its expression
in a given species. To provide optimized polynucleotides coding for
Hendra virus polypeptides, the DNA sequence of the Hendra virus gene can
be modified to 1) comprise codons preferred by highly expressed genes in
a particular species; 2) comprise an A+T or G+C content in nucleotide
base composition to that substantially found in said species; 3) form an
initiation sequence of said species; or 4) eliminate sequences that cause
destabilization, inappropriate polyadenylation, degradation and
termination of RNA, or that form secondary structure hairpins or RNA
splice sites. Increased expression of Hendra virus protein in said
species can be achieved by utilizing the distribution frequency of codon
usage in eukaryotes and prokaryotes, or in a particular species. The term
"frequency of preferred codon usage" refers to the preference exhibited
by a specific host cell in usage of nucleotide codons to specify a given
amino acid. There are 20 natural amino acids, most of which are specified
by more than one codon. Therefore, all degenerate nucleotide sequences
are included in the disclosure as long as the amino acid sequence of the
Hendra virus polypeptide encoded by the nucleotide sequence is
functionally unchanged.

[0060] The sequence identity between two amino acid sequences may be
established by the NCBI (National Center for Biotechnology Information)
pairwise blast and the blosum62 matrix, using the standard parameters
(see, e.g., the BLAST or BLASTX algorithm available on the "National
Center for Biotechnology Information" (NCBI, Bethesda, Md., USA) server,
as well as in Altschul et al.).

[0061] The "identity" with respect to sequences can refer to the number of
positions with identical nucleotides or amino acids divided by the number
of nucleotides or amino acids in the shorter of the two sequences wherein
alignment of the two sequences can be determined in accordance with the
Wilbur and Lipman algorithm (Wilbur and Lipman), for instance, using a
window size of 20 nucleotides, a word length of 4 nucleotides, and a gap
penalty of 4, and computer-assisted analysis and interpretation of the
sequence data including alignment can be conveniently performed using
commercially available programs (e.g., Intelligenetics® Suite,
Intelligenetics Inc. CA). When RNA sequences are said to be similar, or
have a degree of sequence identity or homology with DNA sequences,
thymidine (T) in the DNA sequence is considered equal to uracil (U) in
the RNA sequence. Thus, RNA sequences are within the scope of the
invention and can be derived from DNA sequences, by thymidine (T) in the
DNA sequence being considered equal to uracil (U) in RNA sequences.

[0062] The sequence identity or sequence similarity of two amino acid
sequences, or the sequence identity between two nucleotide sequences can
be determined using Vector NTI software package (Invitrogen, 1600 Faraday
Ave., Carlsbad, Calif.).

[0063] The following documents provide algorithms for comparing the
relative identity or homology of sequences, and additionally or
alternatively with respect to the foregoing, the teachings in these
references can be used for determining percent homology or identity:
Needleman S B and Wunsch C D; Smith T F and Waterman M S; Smith T F,
Waterman M S and Sadler J R; Feng D F and Dolittle R F; Higgins D G and
Sharp P M; Thompson J D, Higgins D G and Gibson T J; and, Devereux J,
Haeberlie P and Smithies O. And, without undue experimentation, the
skilled artisan can consult with many other programs or references for
determining percent homology.

[0064] Hybridization reactions can be performed under conditions of
different stringency. Conditions that increase stringency of a
hybridization reaction are well known. See for example, "Molecular
Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989).

[0065] The invention encompasses the Hendra virus polynucleotide(s)
contained in a vector molecule or an expression vector and operably
linked to a promoter element and optionally to an enhancer.

[0066] The present invention further encompasses a vaccine or composition
which may comprise one or more aforementioned recombinant vector
comprising one or more polynucleotides encoding one or more Hendra virus
polypeptides or antigens, a pharmaceutically or veterinarily acceptable
carrier, excipient, adjuvant, or vehicle. The present invention further
relates to a vaccine or composition which may comprise one or more
aforementioned recombinant or expression vector and additionally one or
more antigens. The additional antigen(s) may be Nipah virus antigen(s).
The antigen may comprise a whole organism, killed, attenuated or live; a
subunit or portion of an organism; a recombinant vector containing an
insert with immunogenic properties; a piece or fragment of DNA capable of
inducing an immune response upon presentation to a host animal; a
polypeptide, an epitope, a hapten, or any combination thereof.

[0067] A "vector" refers to a recombinant DNA or RNA plasmid or virus that
comprises a heterologous polynucleotide to be delivered to a target cell,
either in vitro or in vivo. The heterologous polynucleotide may comprise
a sequence of interest for purposes of prevention or therapy, and may
optionally be in the form of an expression cassette. As used herein, a
vector needs not be capable of replication in the ultimate target cell or
subject. The term includes cloning vectors and viral vectors.

[0068] The term "recombinant" means a polynucleotide with semisynthetic,
or synthetic origin which either does not occur in nature or is linked to
another polynucleotide in an arrangement not found in nature.

[0069] "Heterologous" means derived from a genetically distinct entity
from the rest of the entity to which it is being compared. For example, a
polynucleotide may be placed by genetic engineering techniques into a
plasmid or vector derived from a different source, and is a heterologous
polynucleotide. A promoter removed from its native coding sequence and
operatively linked to a coding sequence other than the native sequence is
a heterologous promoter.

[0070] The polynucleotides of the invention may comprise additional
sequences, such as additional encoding sequences within the same
transcription unit, controlling elements such as promoters, ribosome
binding sites, 5'UTR, 3'UTR, transcription terminators, polyadenylation
sites, additional transcription units under control of the same or a
different promoter, sequences that permit cloning, expression, homologous
recombination, and transformation of a host cell, and any such construct
as may be desirable to provide embodiments of this invention.

[0071] Elements for the expression of a Hendra virus polypeptide, antigen,
epitope or immunogen are present in an inventive vector. In minimum
manner, this comprises an initiation codon (ATG), a stop codon and a
promoter, and optionally also a polyadenylation sequence for certain
vectors such as plasmid and certain viral vectors, e.g., viral vectors
other than poxviruses. When the polynucleotide encodes a polypeptide
fragment, e.g. a Hendra virus polypeptide, in the vector, an ATG is
placed at 5' of the reading frame and a stop codon is placed at 3'. Other
elements for controlling expression may be present, such as enhancer
sequences, stabilizing sequences, such as intron and signal sequences
permitting the secretion of the protein.

[0072] The present invention also relates to compositions or vaccines
comprising vectors.

[0073] The composition or vaccine can comprise one or more vectors, e.g.,
expression vectors, such as in vivo expression vectors, comprising and
expressing one or more Hendra virus polypeptides, antigens, epitopes or
immunogens. In one embodiment, the vector contains and expresses one or
more polynucleotides that comprise one or more polynucleotides coding for
and/or expressing one or more Hendra virus antigen, polypeptide, epitope
or immunogen, in a pharmaceutically or veterinarily acceptable carrier,
excipient, adjuvant, or vehicle.

[0074] According to another embodiment, the vector or vectors in the
composition or vaccine comprise, or consist essentially of, or consist of
polynucleotide(s) encoding one or more proteins or fragment(s) of a
Hendra virus polypeptide, antigen, epitope or immunogen. In another
embodiment, the composition or vaccine comprises one, two, or more
vectors comprising polynucleotides encoding and expressing,
advantageously in vivo, a Hendra virus polypeptide, antigen, fusion
protein or an epitope thereof. The invention is also directed at mixtures
of vectors that comprise polynucleotides encoding and expressing
different Hendra virus polypeptides, antigens, epitopes, fusion protein,
or immunogens, e.g., a Hendra virus F and/or G polypeptide, antigen,
epitope or immunogen from pathogens causing disease in different species
such as, but not limited to, humans, horses, pigs, cows or cattle, dogs,
and cats.

[0076] According to a yet further embodiment of the invention, the
expression vector is a plasmid vector, in particular an in vivo
expression vector. In a specific, non-limiting example, the pVR1020 or
1012 plasmid (VICAL Inc.; Luke et al., 1997; Hartikka et al., 1996, see,
e.g., U.S. Pat. Nos. 5,846,946 and 6,451,769) can be utilized as a vector
for the insertion of a polynucleotide sequence. The pVR1020 plasmid is
derived from pVR1012 and contains the human tPA signal sequence. In one
embodiment the human tPA signal comprises from amino acid M(1) to amino
acid S(23) of GenBank accession number HUMTPA14. In another specific,
non-limiting example, the plasmid utilized as a vector for the insertion
of a polynucleotide sequence can contain the signal peptide sequence of
equine IGF1 from amino acid M(24) to amino acid A(48) of GenBank
accession number U28070. Additional information on DNA plasmids which may
be consulted or employed in the practice are found, for example, in U.S.
Pat. Nos. 6,852,705; 6,818,628; 6,586,412; 6,576,243; 6,558,674;
6,464,984; 6,451,770; 6,376,473 and 6,221,362.

[0077] The term plasmid covers any DNA transcription unit comprising a
polynucleotide according to the invention and the elements necessary for
its in vivo expression in a cell or cells of the desired host or target;
and, in this regard, it is noted that a supercoiled or non-supercoiled,
circular plasmid, as well as a linear form, are intended to be within the
scope of the invention.

[0078] Each plasmid comprises or contains or consists essentially of, in
addition to the polynucleotide(s) encoding the Hendra virus
polypeptide(s), antigen(s), epitopes or immunogens, optionally fused with
a heterologous peptide sequence, variant, analog or fragment, operably
linked to a promoter or under the control of a promoter or dependent upon
a promoter. In general, it is advantageous to employ a strong promoter
functional in eukaryotic cells. The strong promoter may be, but not
limited to, the immediate early cytomegalovirus promoter (CMV-IE) of
human or murine origin, or optionally having another origin such as the
rat or guinea pig.

[0079] In more general terms, the promoter has either a viral, or a
cellular origin. A strong viral promoter other than CMV-IE that may be
usefully employed in the practice of the invention is the early/late
promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus.
A strong cellular promoter that may be usefully employed in the practice
of the invention is the promoter of a gene of the cytoskeleton, such as
e.g. the desmin promoter (Kwissa et al., 2000), or the actin promoter
(Miyazaki et al., 1989).

[0080] As to the polyadenylation signal (polyA) for the plasmids and viral
vectors other than poxviruses, use can be made of the poly(A) signal of
the bovine growth hormone (bGH) gene (see U.S. Pat. No. 5,122,458), or
the poly(A) signal of the rabbit β-globin gene or the poly(A) signal
of the SV40 virus.

[0081] A "host cell" denotes a prokaryotic or eukaryotic cell that has
been genetically altered, or is capable of being genetically altered by
administration of an exogenous polynucleotide, such as a recombinant
plasmid or vector. When referring to genetically altered cells, the term
refers both to the originally altered cell and to the progeny thereof.

Methods of use and Article of Manufacture

[0082] The present invention includes the following method embodiments. In
an embodiment, a method of vaccinating an animal comprising administering
composition comprising a vector comprising one or more polynucleotides
encoding one or more Hendra virus polypeptides or fragments or variants
thereof and a pharmaceutical or veterinarily acceptable carrier,
excipient, vehicle, or adjuvant to an animal and human is disclosed. In
one aspect of this embodiment, the animal is an equine, a canine, a
feline, or a porcine.

[0083] In yet another embodiment, a method of vaccinating an animal
comprising a composition comprising one or more vectors comprising one or
more polynucleotides encoding one or more Hendra virus polypeptides and
optionally a pharmaceutical or veterinarily acceptable carrier,
excipient, vehicle, or adjuvant and optionally one or more compositions
comprising additional antigens is disclosed.

[0084] In one embodiment of the invention, a prime-boost regimen can be
employed, which is comprised of at least one primary administration and
at least one booster administration using at least one common
polypeptide, antigen, epitope or immunogen. The administration may
comprise one, two, or more vaccines or compositions comprising same or
different antigens. Typically the immunological composition(s) or
vaccine(s) used in primary administration is different in nature from
those used as a booster. However, it is noted that the same
composition(s) can be used as the primary administration and the booster
administration. This administration protocol is called "prime-boost".

[0085] A prime-boost regimen comprises at least one prime-administration
and at least one boost administration using at least one common
polypeptide and/or variants or fragments thereof. The
prime-administration may comprise one or more administrations. Similarly,
the boost administration may comprise one or more administrations. The
prime-administration may comprise one or more antigens and the boost
administration may comprise one or more antigens.

[0086] In one aspect of the prime-boost protocol or regime of the
invention, a prime-boost protocol may comprise the administration of a
composition comprising a recombinant viral vector that contains and
expresses one or more Hendra virus polypeptides, antigens and/or variants
or fragments thereof in vivo followed by the administration of one or
more recombinant Hendra virus polypeptides or antigens, or an inactivated
viral composition or vaccine comprising the Hendra virus polypeptides or
antigens, or a DNA plasmid-based composition or vaccine expressing one or
more Hendra virus polypeptides or antigens. Likewise, a prime-boost
protocol may comprise the administration of a composition comprising one
or more recombinant Hendra virus antigens, or an inactivated viral
composition or vaccine comprising the Hendra virus polypeptides or
antigens, or a DNA plasmid-based composition or vaccine expressing the
Hendra virus polypeptide or antigen followed by the administration of a
recombinant viral vector that contains and expresses one or more Hendra
virus polypeptides or antigens and/or variants or fragments thereof in
vivo. It is further noted that both the primary and the secondary
administrations may comprise the recombinant viral vector that contains
and expresses one or more Hendra virus polypeptides of the invention.
Thus, the recombinant Hendra viral vector of the invention may be
administered in any order with one or more recombinant Hendra virus
antigens, an inactivated viral composition or vaccine comprising the
Hendra virus antigens, or a DNA plasmid-based composition or vaccine
expressing one or more Hendra virus antigens, or alternatively may be
used alone as both the primary and secondary compositions.

[0087] The dose volume of compositions for target species that are
mammals, e.g., the dose volume of dog compositions, based on viral
vectors, e.g., non-poxvirus-viral-vector-based compositions, is generally
between about 0.1 to about 2.0 ml, between about 0.1 to about 1.0 ml, and
between about 0.5 ml to about 1.0 ml.

[0088] The efficacy of the vaccines may be tested about 2 to 4 weeks after
the last immunization by challenging animals, such as horses, cats, dogs,
pigs, or experimental laboratory animals (such as ferrets and guinea
pigs) with a virulent strain of Hendra virus strain. Both homologous and
heterologous strains are used for challenge to test the efficacy of the
vaccine. The animal may be challenged by spray, intra-nasally,
intra-ocularly, intra-tracheally, and/or orally. The challenge viral may
be about 105-8 EID50 in a volume depending upon the route of
administration. For example, if the administration is by spray, a virus
suspension is aerosolized to generate about 1 to 100 μm droplets, if
the administration is intra-nasal, intra-tracheal or oral, the volume of
the challenge virus is about 0.5 ml, 1-2 ml, and 5-10 ml, respectively.
Animals may be observed daily for 14 days following challenge for
clinical signs, for example, dehydration and fever. In addition, the
groups of animals may be euthanized and evaluated for pathological
findings of pulmonary and pleural hemorrhage, tracheitis, bronchitis,
bronchiolitis, bronchopneumonia and internal organs. Orophayngeal swabs
may be collected from all animals post challenge for virus isolation. The
presence or absence of viral antigens in respiratory tissues may be
evaluated by quantitative real time reverse transcriptase polymerase
chain reaction (qRT-PCR). Blood samples may be collected before and
post-challenge and may be analyzed for the presence of Hendra
virus-specific antibody.

[0089] The various administrations are preferably carried out 1 to 6 weeks
apart. Preferred time interval is 3 to 5 weeks, and optimally 4 weeks.
According to one embodiment, a six-month booster interval or an annual
booster interval is also envisioned. The animals, for examples horses,
may be at least four months of age at the time of the first
administration.

[0090] It should be understood by one of skill in the art that the
disclosure herein is provided by way of example and the present invention
is not limited thereto. From the disclosure herein and the knowledge in
the art, the skilled artisan can determine the number of administrations,
the administration route, and the doses to be used for each injection
protocol, without any undue experimentation.

[0091] The present invention contemplates at least one administration to
an animal of an efficient amount of the therapeutic composition made
according to the invention. The animal may be male, female, pregnant
female and newborn. This administration may be via various routes
including, but not limited to, intramuscular (IM), intradermal (ID) or
subcutaneous (SC) injection or via intranasal or oral administration. The
therapeutic composition according to the invention can also be
administered by a needleless apparatus (as, for example with a Pigjet,
Dermojet, Biojector, Avijet (Merial, GA, USA), Vet et or Vitajet
apparatus (Bioject, Oregon, USA)). Another approach to administering
plasmid compositions is to use electroporation (see, e.g. Tollefsen et
al., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT Application
No. WO99/01158). In another embodiment, the therapeutic composition is
delivered to the animal by gene gun or gold particle bombardment.

[0092] The recombinant composition or vaccine can be administered to an
animal or infected or transfected into cells in an amount of about 1.0
log 10 TCID50 (or CCID50) to about 20.0 log 10 TCID50 (or CCID50), about
1.0 log 10 TCID50 (or CCID50) to about 15.0 log 10 TCID50 (or CCID50),
about 2.0 log 10 TCID50 (or CCID50) to about 10.0 log 10 TCID50 (or
CCID50), or about 4.0 log 10 TCID50 (or CCID50) to about 8.0 log 10
TCID50 (or CCID50).

[0093] In one embodiment, the invention provides for the administration of
a therapeutically effective amount of a formulation for the delivery and
expression of a Hendra virus antigen or epitope in a target cell.
Determination of the therapeutically effective amount is routine
experimentation for one of ordinary skill in the art. In one embodiment,
the formulation comprises an expression vector comprising a
polynucleotide that expresses one or more Hendra virus antigens or
epitopes and a pharmaceutically or veterinarily acceptable carrier,
vehicle, adjuvant, or excipient.

[0094] The pharmaceutically or veterinarily acceptable carriers or
vehicles or excipients or adjuvants are well known to the one skilled in
the art. For example, a pharmaceutically or veterinarily acceptable
carrier or vehicle or excipient or adjuvant can be a 0.9% NaCl (e.g.,
saline) solution or a phosphate buffer. Other pharmaceutically or
veterinarily acceptable carrier or vehicle or excipient or adjuvant that
can be used for methods of this invention include, but are not limited
to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or
veterinarily acceptable carrier or vehicle or excipient or adjuvant may
be any compound or combination of compounds facilitating the
administration of the vector (or protein expressed from an inventive
vector in vitro) and the transfection or infection and/or improves
preservation of the vector or protein in a host. Doses and dose volumes
are herein discussed in the general description and can also be
determined by the skilled artisan from this disclosure read in
conjunction with the knowledge in the art, without any undue
experimentation.

[0095] The cationic lipids containing a quaternary ammonium salt which are
advantageously but not exclusively suitable for plasmids, are those
having the following formula:

##STR00001##

[0096] in which R1 is a saturated or unsaturated straight-chain aliphatic
radical having 12 to 18 carbon atoms, R2 is another aliphatic radical
containing 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g.
the DMRIE. In another embodiment the cationic lipid can be associated
with a neutral lipid, e.g. the DOPE.

[0097] Among these cationic lipids, preference is given to DMRIE
(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane
ammonium; WO96/34109), advantageously associated with a neutral lipid,
advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr, 1994), to
form DMRIE-DOPE.

[0098] When DOPE is present, the DMRIE:DOPE molar ratio is advantageously
about 95:about 5 to about 5:about 95, more advantageously about 1:about
1, e.g., 1:1.

[0099] In another embodiment, pharmaceutically or veterinarily acceptable
carrier, excipient, vehicle or adjuvant may be a water-in-oil emulsion.
Examples of suitable water-in-oil emulsions include oil-based
water-in-oil vaccinal emulsions which are stable and fluid at 4°
C. containing: from 6 to 50 v/v % of an antigen-containing aqueous phase,
preferably from 12 to 25 v/v %, from 50 to 94 v/v % of an oil phase
containing in total or in part a non-metabolizable oil (e.g., mineral oil
such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil, or
fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % of
surfactants, preferably from 3 to 8 p/v %, the latter being in total or
in part, or in a mixture either polyglycerol esters, said polyglycerol
esters being preferably polyglycerol (poly)ricinoleates, or
polyoxyethylene ricin oils or else hydrogenated polyoxyethylene ricin
oils. Examples of surfactants that may be used in a water-in-oil emulsion
include ethoxylated sorbitan esters (e.g., polyoxyethylene (20) sorbitan
monooleate (TWEEN 80®), available from AppliChem, Inc., Cheshire,
Conn.) and sorbitan esters (e.g., sorbitan monooleate (SPAN 80®),
available from Sigma Aldrich, St. Louis, Mo.). In addition, with respect
to a water-in-oil emulsion, see also U.S. Pat. No. 6,919,084. In some
embodiments, the antigen-containing aqueous phase comprises a saline
solution comprising one or more buffering agents. An example of a
suitable buffering solution is phosphate buffered saline. In one
embodiment, the water-in-oil emulsion may be a water/oil/water (W/O/W)
triple emulsion (U.S. Pat. No. 6,358,500). Examples of other suitable
emulsions are described in U.S. Pat. No. 7,371,395.

[0100] The immunological compositions and vaccines according to the
invention may comprise or consist essentially of one or more
pharmaceutically or veterinarily acceptable carriers, excipients,
vehicles or adjuvants. Suitable adjuvants for use in the practice of the
present invention are (1) polymers of acrylic or methacrylic acid, maleic
anhydride and alkenyl derivative polymers, (2) immunostimulating
sequences (ISS), such as oligodeoxyribonucleotide sequences having one or
more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an
oil in water emulsion, such as the SPT emulsion described on page 147 of
"Vaccine Design, The Subunit and Adjuvant Approach" published by M.
Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on
page 183 of the same work, (4) cation lipids containing a quaternary
ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or
aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any
document cited and incorporated by reference into the instant
application, or (9) any combinations or mixtures thereof.

[0101] The oil in water emulsion (3), which is especially appropriate for
viral vectors, can be based on: light liquid paraffin oil (European
pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil
resulting from the oligomerization of alkenes, e.g. isobutene or decene,
esters of acids or alcohols having a straight-chain alkyl group, such as
vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate),
glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters
of branched, fatty alcohols or acids, especially isostearic acid esters.

[0102] The oil is used in combination with emulsifiers to form an
emulsion. The emulsifiers may be nonionic surfactants, such as: esters of
on the one hand sorbitan, mannide (e.g. anhydromannitol oleate),
glycerol, polyglycerol or propylene glycol and on the other hand oleic,
isostearic, ricinoleic or hydroxystearic acids, said esters being
optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer
blocks, such as Pluronic, e.g., L121.

[0103] Among the type (1) adjuvant polymers, preference is given to
polymers of crosslinked acrylic or methacrylic acid, especially
crosslinked by polyalkenyl ethers of sugars or polyalcohols. These
compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2,
June 1996). One skilled in the art can also refer to U.S. Pat. No.
2,909,462, which provides such acrylic polymers crosslinked by a
polyhydroxyl compound having at least three hydroxyl groups, preferably
no more than eight such groups, the hydrogen atoms of at least three
hydroxyl groups being replaced by unsaturated, aliphatic radicals having
at least two carbon atoms. The preferred radicals are those containing 2
to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically
unsaturated groups. The unsaturated radicals can also contain other
substituents, such as methyl. Products sold under the name Carbopol (BF
Goodrich, Ohio, USA) are especially suitable. They are crosslinked by
allyl saccharose or by allyl pentaerythritol. Among them, reference is
made to Carbopol 974P, 934P and 971P.

[0104] As to the maleic anhydride-alkenyl derivative copolymers,
preference is given to EMA (Monsanto), which are straight-chain or
crosslinked ethylene-maleic anhydride copolymers and they are, for
example, crosslinked by divinyl ether.

[0105] With regard to structure, the acrylic or methacrylic acid polymers
and EMA are preferably formed by basic units having the following
formula:

##STR00002##

in which: [0106] R1 and R2, which can be the same or different,
represent H or CH3 [0107] x=0 or 1, preferably x=1 [0108] y=1 or 2, with
x+y=2.

[0109] For EMA, x=0 and y=2 and for carbomers x=y=1.

[0110] These polymers are soluble in water or physiological salt solution
(20 g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda
(NaOH), to provide the adjuvant solution in which the expression
vector(s) can be incorporated. The polymer concentration in the final
immunological or vaccine composition can range between about 0.01 to
about 1.5% w/v, about 0.05 to about 1% w/v, and about 0.1 to about 0.4%
w/v.

[0111] The cytokine or cytokines (5) can be in protein form in the
immunological or vaccine composition, or can be co-expressed in the host
with the immunogen or immunogens or epitope(s) thereof. Preference is
given to the co-expression of the cytokine or cytokines, either by the
same vector as that expressing the immunogen or immunogens or epitope(s)
thereof, or by a separate vector thereof.

[0112] The invention comprehends preparing such combination compositions;
for instance by admixing the active components, advantageously together
and with an adjuvant, carrier, cytokine, and/or diluent.

[0113] Cytokines that may be used in the present invention include, but
are not limited to, granulocyte colony stimulating factor (G-CSF),
granulocyte/macrophage colony stimulating factor (GM-CSF), interferon
α (IFNα), interferon β (IFNβ), interferon γ,
(IFNγ), interleukin-1α (IL-1α), interleukin-1β
(IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4
(IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),
interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10),
interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor
α (TNFα), tumor necrosis factor β (TNFβ), OX40L,
and transforming growth factor β (TGFβ). It is understood that
cytokines can be co-administered and/or sequentially administered with
the immunological or vaccine composition of the present invention. Thus,
for instance, the vaccine of the instant invention can also contain an
exogenous nucleic acid molecule that expresses in vivo a suitable
cytokine, e.g., a cytokine matched to this host to be vaccinated or in
which an immunological response is to be elicited (for instance, a canine
cytokine for preparations to be administered to canine).

[0114] The invention will now be further described by way of the following
non-limiting examples.

EXAMPLES

[0115] Without further elaboration, it is believed that one skilled in the
art can, using the preceding descriptions, practice the present invention
to its fullest extent. The following detailed examples are to be
construed as merely illustrative, and not limitations of the preceding
disclosure in any way whatsoever. Those skilled in the art will promptly
recognize appropriate variations from the procedures both as to reactants
and as to reaction conditions and techniques.

[0120] The IVR (in vitro recombinant) was performed by transfection of
Primary chicken embryo fibroblast (1°CEF) cells with NotI
linearized donor plasmid p362-Hendra G. The transfected cells were
subsequently infected with parental ALVAC as rescue virus at MOI
(multiplicity of infection) of 10. After 24 hours, the
transfected/infected cells were harvested, sonicated and used for
recombinant virus screening. Recombinant plaques were screened based on
the plaque lift hybridization method using Hendra G-specific probe which
was labeled with horse radish peroxidase according to the manufacturer's
protocol (GE Healthcare, Cat #RPN3001). After four sequential rounds of
plaque purification, the recombinants designated as vCP3004.1.1.1.1. and
vCP3004.5.3.2.2 were generated and confirmed by hybridization as 100%
positive for the Hendra G insert and 100% negative for the C5 ORF.

B. Genomic Analysis

[0121] Genomic DNA from vCP3004.1.1.1.1 was extracted and digested with
BamHI, HindIII and PstI, separated by agarose electrophoresis and then
transferred to nylon membrane. Southern blot was performed by probing
with a Hendra G probe. The primers used to generate the Hendra G probe
are:

[0123] Primary CEF cells were infected with vCP3004.1.1.1.1 at MOI of 10
and incubated at 37° C. for 24 hours. The cells and culture
supernatant were then harvested. Sample proteins were separated on a 10%
SDS-PAGE gel, transferred to PVDF membrane. A serum raised in guinea pig
reacted strongly with the G protein at an apparent molecular size of
approximately 70 kDa. The result is shown in FIG. 4.

D. Sequence Analysis

[0124] A more detailed analysis of the P3 stock genomic DNA was performed
by PCR amplification and sequence analysis of the flanking arms of the C5
locus and the Hendra G insert. Primers C5R.1F and C5L.2R located at the
end of the arms of the C5 locus in the donor plasmid were used to amplify
the entire C5R-Hendra G insert-C5L fragment.

[0127] The IVR (in vitro recombinant) was performed by transfection of
Primary chicken embryo fibroblast (1°CEF) cells with NotI
linearized donor plasmid p362-Hendra F. The transfected cells were
subsequently infected with parental ALVAC as rescue virus at MOI
(multiplicity of infection) of 10. After 24 hours, the
transfected/infected cells were harvested, sonicated and used for
recombinant virus screening. Recombinant plaques were screened based on
the plaque lift hybridization method using Hendra F-specific probe which
was labelled with horse radish peroxidase according to the manufacturer's
protocol (GE Healthcare, Cat #RPN3001). After four sequential rounds of
plaque purification, the recombinants designated as vCP3005.3.4.1 and
vCP3005.5.3.2 were generated and confirmed by hybridization as 100%
positive for the Hendra F insert and 100% negative for the C5 ORF.

B. Genomic Analysis

[0128] Genomic DNA from vCP3005.3.4.1 was extracted and digested with
BamHI, HindIII and PstI, separated by agarose electrophoresis and then
transferred to nylon membrane. Southern blot was performed by probing
with a Hendra F probe. The primers used to generate the Hendra F probe
are:

[0130] Primary CEF cells were infected with vCP3005.3.4.1 at MOI of 10 and
incubated at 37° C. for 24 hours. The cells and culture
supernatant were then harvested. Sample proteins were separated on a 10%
SDS-PAGE gel, transferred to PVDF membrane. When a serum raised in guinea
pig was used in the Western blot, a faint band corresponding to uncleaved
F0 protein at approximately 60 kDa was recognized. The result is shown in
FIG. 6.

D. Sequence Analysis

[0131] A more detailed analysis of the P3 stock genomic DNA was performed
by PCR amplification and sequence analysis of the flanking arms of the C5
locus and the Hendra F insert. Primers C5R.1F (SEQ ID NO:15) and C5L.2R
(SEQ ID NO:16) located at the end of the arms of the C5 locus in the
donor plasmid were used to amplify the entire C5R-Hendra F inset-C5L
fragment.

Example 5

Fusion Assay

[0132] Simultaneous co-infection of HEK293 cells with the ALVAC-Hendra G
(vCP3004) and ALVAC-Hendra F (vCP3005) at an MOI of 10+10 resulted in
syncytium formation, while single infections either ALVAC-Hendra F
(vCP3005) or ALVAC-Hendra G (vCP3004) recombinant virus at MOI of 20 did
not result in syncytium formation, demonstrating the functionality of
both proteins (see FIG. 7).

Example 6

Serology Study of Horses Vaccinated with ALVAC-Hendra F or G and
ALVAC-Nipah F or G

[0134] In this study, two groups of horses were vaccinated IM with the
mixture of vCP-Hendra G vector (vCP3004) and vCP-Nipah F vector (ALVAC
vector containing Nipah F) on D0 and D28. Group 1 received the vector
mixture in Carbomer at 5.8 log 10 TCID50/dose. Group 2 received the
vector mixture in Carbomer at 6.8 log 10 TCID50/dose. Sera were titrated
for antibodies against Hendra G and F proteins and Nipah G and F proteins
in serum neutralization titre (SNT) test. Sera were also tested in ELISA
blocking and binding assays using antibodies against Hendra G protein and
Nipah G protein respectively.

[0135] FIGS. 8A-C show the ELISA binding assay and blocking assay using
antibodies against Hendra G protein, and SNT test against Hendra G and F
proteins. FIGS. 9A-C show the binding assay and blocking assay using
antibodies against Nipah G protein, and SNT test against Nipah G and F
proteins.

[0136] The results showed that vaccination of horses with vCP-Hendra G
vector and vCP-Nipah F vector induced anti-Hendra and anti-Nipah
responses even as late as D70.

[0138] The clinical result showed that vaccinations are safe for both
groups. There is no difference between groups 1 and 2. Biodiffusibility
data showed that no virus was detected in any samples.

[0139] FIG. 10A shows the virus neutralization (VN) test against Hendra.
Both groups showed above the theoretical protection threshold (64 titre)
from D70 onward up to D155. After the third injection on D183, both
groups showed clear booster effect.

[0140] FIG. 10B shows the VN test against Nipah. The results showed good
cross reactivity against Nipah. Most horses showed above the protection
threshold (60 titre) after the third injection on D183, and some horses
showed some protection even after the second injection on D28.

[0142] No clinical signs were reported in any one of the four groups.
There is no difference in histology between the vaccinated groups.

[0143] On D8, virus was detected on the skin of all canaries vaccinated
with CPpp (ranging from 2.79 to 6.65 log10 CCID50/ml) and all
but one canaries vaccinated with vCP3004 (ranging from 3.22 to 6.80
log10 CCID50/m1). On D16, no virus was detected in any
vaccinated groups.

[0144] Sampling of the pool of organs showed that no virus was detected in
any canaries in the two inoculated groups and the two contact groups on
D8 and D16.

[0145] The results demonstrated the safety and the absence of spreading of
vCP3004 administered at high titre by transcutaneous route to the canary.
The absence of reactions and virus isolation in the sentinel canaries
confirmed the absence of spread of vCP3004 in this species.

[0147] The result showed that there was no clinical sign for any
vaccinated group. On D8 and D16, after the first passage, no virus could
be isolated from the organ samples in both inoculated groups and contact
animals. This study demonstrated the safety and the absence of spreading
of vCP3005 administered at high titre by transcutaneous route to the
canary. The absence of reactions and virus isolation in the sentinel
canaries confirmed the absence of spread of vCP3005 in this species.

[0148] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention defined by
the above paragraphs is not to be limited to particular details set forth
in the above description as many apparent variations thereof are possible
without departing from the spirit or scope of the present invention.

[0149] All documents cited or referenced in the application cited
documents, and all documents cited or referenced herein ("herein cited
documents"), and all documents cited or referenced in herein cited
documents, together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products mentioned
herein or in any document incorporated by reference herein, are hereby
incorporated herein by reference, and may be employed in the practice of
the invention.